Dynamic bh3 profiling

ABSTRACT

The present invention provides methods of predicting cell sensitivity or resistance to a therapeutic agent.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/429,272, filed Mar. 18, 2015, which is a national stage filing under35 U.S.C. § 371 of International Application No. PCT/US2013/060707,filed Sep. 19, 2013, which claims priority to and the benefit ofprovisional application U.S. Ser. No. 61/702,967, filed on Sep. 19,2012, the contents of each of which are incorporated herein by referencein their entirety.

FIELD OF INVENTION

The invention relates to generally to methods of predicting response tochemotherapy and in particular targeted therapies.

BACKGROUND OF THE INVENTION

As more targeted therapies are approved for different types of cancer,there is a growing need for predictive biomarkers so that thesetherapies can be directed to patients who will most benefit from them;unfortunately, the biomarkers available for cancer therapy are notsufficient. Recently, the development of tyrosine kinase inhibitors(TKI) improved treatment in patients with advanced disease. For example,detection of mutations in EGFR has been successfully used as a biomarkerfor initial therapy with EGFR inhibitors. Many targeted agents lackgenetic predictive biomarkers. Furthermore, resistance to these drugsfrequently emerges, and it is often not clear what treatment is bestgiven following this emergence of resistance, given the variety ofmechanisms for resistance. The present invention provides a method ofpredicting the response to therapy so that drugs can better be assignedto patients.

SUMMARY OF THE INVENTION

In various aspects, the invention provides methods of predicting theresponse to chemotherapy.

In various aspects the invention provides methods of predictingsensitivity of a cell to a therapeutic agent by contacting a test cellpopulation that has been exposed to a test therapeutic agent with apro-apoptotic BH3 domain peptide, measuring the amount of BH3 domainpeptide induced mitochondrial outer membrane permeabilization in thetest cell population and comparing the amount of BH3 domain peptideinduced mitochondrial outer membrane membrane permeabilization in thetest cell population to a control cell population that has not beencontacted with the therapeutic agent. An increase in mitochondrialsensitivity to a BH3 domain peptide in the test cell population comparedto the control cell population indicates the cell is sensitive to thetherapeutic agent. In various embodiments the cell is permeabilizedprior to contacting with the BH3 domain peptide. In various aspects, themethod further comprises contacting the permeabilized cell with apotentiometric dye. Potentiometric dyes include for example JC-1 ordihydrorhodamine 123.

Mitochondrial outer membrane membrane permeabilization is determined forexample by measuring i) the emission of a potentiometric or ratiometricdye or ii) the release of molecules from the mitochondrialinter-membrane space.

BH3 domain peptides include peptides derived from the BH3 domain of aBID, a BIM, a BAD, a NOXA, a PUMA, a BMF, or a HRK polypeptide.Exemplary BH3 domain peptides include SEQ ID NO: 1-14

The therapeutic agent is a chemotherapeutic agent such as a kinaseinhibitor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show dynamic BH3 profiling in NSCLC cell lines PC9, PC9GRand PC9WZR. FIG. 1A shows the BH3 profiling results on cells exposed todrug for 16 h, using mitochondrial response to the Bim peptide (0.3 μM)response to measure priming. FIG. 1B shows cell death was measured at 72h by FACS using Annexin V/PI staining.

FIGS. 2A-2C show that mutant BIM AV peptide works like original BIMpeptide. Dynamic BH3 profiling in cell lines PC9 (FIG. 2A), PC9GR (FIG.2B) and PC9WZR (FIG. 2C) exposed to gefitinib 1 μM or WZ4002 100 nM for16 h, using BIM BH3 peptide Ac-MRPEIWIAQELRRIGDEFNA-NH2 (SEQ ID NO: 1)(0.3 μM or 1 μM) or point-mutated BIM AV BH3 peptideAc-MRPEIWIAQELRRIGDEFNV-NH2 (SEQ ID NO: 2) (0.3 μM or 1 μM) response tomeasure priming.

FIGS. 3A-3C show dynamic BH3 profiling in NSCLC cell lines. FIG. 3Ashows BH3 profiling results on cells exposed to drug for 16 h, using Bimpeptide (1 μM) response to measure priming. FIG. 3B shows cell death wasmeasured at 72 h by FACS using Annexin V/PI staining. FIG. 3C shows thecorrelation between Δ % depolarization with Bim 1 μM and Cell Death at72 h, p=0.0014; two-tailed.

FIGS. 4A-4D show the BH3 profiling results on human CML cell lines K562(FIG. 4A) and Ku812 (FIG. 4B) treated for 16 h and 8 h with imatinib 1μM, using several BH3 peptides. FIGS. 4C-4D show the correlation betweenΔ % depolarization with Bim 0.1 μM (FIG. 4C) and cell death at 48 h byFACS using Annexin V/PI staining (FIG. 4D).

FIGS. 5A-5B show that dynamic BH3 profiling (DBP) predicts imatinibresponse in CML primary samples. DBP predicting capacity in ChronicMyelogenous Leukemia patient samples were tested. FIG. 5A shows frozenFicoll purified Bone Marrow primary CML samples were treated for 16 hourwith imatinib 1 and 5 μM, and DBP was then performed. Results areexpressed as Δ % priming. Those samples obtained from patients thatresponded to imatinib treatment in clcinc, showed a significantlyhighter Δ % priming in our DBP analysis, as opposed to those samplesobtained form patients that relapsed. FIG. 5B shows a Receiver OperatingCharacteristic curve analysis for this set of samples was performed. Thearea under the ROC curve is 0.94, indivicating the DBP could be used asbinary predictor for CML patients to predict if they will benefit fromimatinib treatment.

FIGS. 6A-6F show that dynamic BH3 profiling accurately predicts leukemiacell death response to targeted therapies. FIG. 6A shows K562 myeloidleukemia cells were exposed to a panel of inhibitors of a range ofkinases for 16 hours, and dynamic BH3 profiling was performed. Thechange in depolarization caused by the BIM BH3 peptide following drugtreatment is shown. Note that significant changes were found only forimatinib (BCR-Abl inhibitor), TAE-684 (ALK), and BEZ235 (PI3K/mTOR).FIG. 6B shows K562 cells were exposed to the same panel of drugs for 72hours and the cell death response was evaluated by Annexin V/PI. Notethat dynamic BH3 profiling accurately predicted the expected killing byimatinib, but also the unexpected killing by TAE-684 and BEZ235. FIG. 6Cshows that dynamic BH3 profiling of BaF3 murine leukemia cells with andwithout p210 reveals differential priming changes induced by differentdrugs after 16 hour exposure. FIG. 6D shows cytotoxicity (Annexin V/PI)after 48 hours exposure confirms prediction of Dynamic BH3 profiling.FIG. 6E shows the dynamic BH3 profiling of Ku812 AML cells exposed toepigenetic modifying agents (from Project 4). Imatinib is a positivecontrol. BET inhibitor JQ1, but not compounds A (DOT1L inhibitor) and B(EZH2 inhibitor), increases priming and is predicted to cause celldeath. FIG. 6F shows Imatinib and JQ1, but not compounds A and B, inducecell death as predicted by dynamic BH3 profiling.

FIGS. 7A-7C show identification of the optimal treatment inhematological malignancies using DBP. Several drugs targeting either keymembrane receptors: geftinib (EGFR inh), imatinib (Abl inh.), lapatinib(HER2 inh.), PD173074 (FGFR inh.) and TAE-684 (Alk inh.); or importantintracellular kinases: MK-2206 (Akt inh.), PLX-4032 (Braf^(V600E)inh.),AZD-6244 (MEK inh.) and BEZ-235 (PI3K/mTOR inh.) were selected, and theywere tested in several human hematologicla cancer cell lines: K562(Chronic Myelogenous leukemia), DHL6 (Diffuse large B-cell lymphoma),LP1 (Multiple Myeloma), DHL4 (Diffuse large B-cell lymphoma) and AML3(Acute Myeloid Leukemia). FIG. 7A shows DBP (16 hour incubation) resultsexpressed as Δ % priming. FIG. 7B shows cell death measurements at 72hours using Annexin V/PI staining expressed as Δ % Cell Death. FIG. 7Cshows a significant correlation between Δ % priming and Δ % Cell Death.Therefore, DBP can predict the optimal treatment for hematologicalmalignancies' cell lines.

FIGS. 8A-8C show identification of the optimal treatment in solid tumorsusing DBP. The same panel of kinase inhibitors used in FIGS. 7A-7C weretested on several human solid tumor cell lines: MCF7 (Breast Cancer),PC9 (Non-Small Cell Lung Cancer), Sk5mel (Melanoma), HCT116 (Coloncarcinoma) and MDA-MB-231 (Breast Cancer). FIG. 8A shows DBP (16 hourincubation) results expressed as Δ % priming. FIG. 8B shows cell deathmeasurements at 72-96 hours using Annexin V/PI staining expressed as Δ %Cell Death. FIG. 8C shows a significant correlation between Δ % primingand Δ % Cell Death. Therefore, DBP can also predict the optimaltreatment for solid tumors' cell lines.

FIGS. 9A-9B show that dynamic BH3 profiling is a good binary predictor.FIG. 9A shows a compilation of FIGS. 7A-7C and FIGS. 8A-8C results,showing a significant correlation between Δ % priming and Δ % Cell Deathfor all the cell lines. FIG. 9B shows a Receiver OperatingCharacteristic curve analysis. The area under the ROC curve is 0.87,indicating that is a good binary predictor for chemotherapy response incell lines.

FIG. 10 is a schematic illustrating the methods of the invention.

FIGS. 11A-11B are a series of bar graphs demonstrating that iBH3 canreproduce the profile of individual subpopulations with mixedpopulations. Samples profiled individually (unmixed as shown in FIG.11A) or as a complex mixture (mixed as shown in FIG. 11B) produce thesame profile.

FIGS. 12A-12B are a series of panels showing how iBH3 definites cellpopulations and measures cellular response to profiling. RepresentativeFACS data (FIGS. 12A and 12B) demonstrates the isolation ofsubpopulations within the mixed sample in FIG. 11B.

FIG. 13 is a series of fluorescent microscopy images that show the lossof cytochrome c in response to peptide treatment measured by microscopy.Cells are located by DAPI staining of their nuclei, mitochondria arelocated by staining of a mitochondrial marker (MnSOD) adjacent tonuclei, and cytochrome c staining is correlated with regions ofmitochondrial marker staining. An inert control peptide shows cytochromec staining in regions of MnSOD staining while BIM peptide causes almosttotal loss of cytochrome c from all regions of MnSOD staining.

FIGS. 14A-14B are a series of bar graphs showing the correlation ofmiBH3 profiles with known profiles. The miBH3 profile of the SuDHL4 cellline (FIG. 14A) shows loss of correlation between cytochrome c and MnSODchannels in response to BH3 peptides. Release of cytochrome c and lossof correlation for BIM, BAD, PUMA, and BMF peptides match the loss ofcytochrome c measured by other BH3 profiling methods shown in FIG. 14B.

FIG. 15 is a graph showing that pre-made frozen plates perform the sameas freshly prepared plates. Responsive cells (MDA-MB-231) showcomparable response to a peptide treatment (BAD) in both frozen andfreshly prepared plates. Non-responsive cells (SuDHL10) are used to testfor non-specific noise, and frozen plates produce a response equivalentto freshly prepared plates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery of a techniquethat measures how close a cancer cell is to the threshold of programmedcell death (i.e. apoptosis), also know as measuring how “primed” thecancer cell is for death. The methods of the invention allow theidentification of drugs that move cancer cells closer to the thresholdof programmed cell death (increase priming the most). This invention canbe applied to individual clinical cancer samples, so that those drugsthat move the cells in that sample closest to the threshold ofprogrammed cell death for that individual sample can be readilyidentified. The drugs so identified are those most likely to provideclinical benefit to the subject from which the sample was derived.Therefore the invention provides a method of personalizing therapy forindividual cancer patients.

This technique differs from previous techniques described inUS2008/0199890 in that the method of the present invention allows forthe observation of the dynamic effects of any number of individual drugsor combination thereof on the mitochondrial priming of an individualcancer sample. The previous method solely measured the priming of acancer sample at baseline, unperturbed by any panel of chemical agents.Those cells that were closest to the apoptotic threshold were thereforemost primed for death and were predicted to be most responsive tochemotherapy generally, but without the ability to discriminate to whichagent a cell was likely to be most sensitive. In contrast, the method ofthis invention allows the change in priming attributed to a particulardrug to be assessed, thus determining whether the compound causes thecell to move closer to the apoptotic threshold. Moreover, the methods ofthe invention are superior to previous methods as it is capable ofdetermining whether a particular cell has become resistant to aparticular therepatic agent. The methods of the invention are referredto herein as Dynamic BH3 Profiling.

Dynamic BH3 Profiling

In various methods, sensitivity of a cell to an agent is determined. Themethods include contacting a test cell with a test agent. Cellsensitivity to the test agent is determined by contacting the test cellor test cellular component (e.g., mitochondria) exposed to the testagent with standardized concentration of a panel of BH3 domain peptidefrom the pro-apoptotic BCL-2 family. Pro-apoptotic BCL-2 BH3 peptidesproteins include: Bcl-2 interacting mediator of cell death (BIM); amutant thereof (BIM AV); BH3 interacting domain death agonist (BID);Bcl-2-associated death promoter (BAD); NOXA; p53 up-regulated modulatorof apoptosis (PUMA); Bcl-2-modifying factor (BMF) and harakiri (HRK)(See, Table 1). The ability of BH3 peptides to induce mitochondrialouter membrane permeabilization is measured in the the test population(i.e. cell or cellular component (e.g., mitochondria) and the controlpopulation (i.e. cell or cellular component (e.g., mitochondria) notexposed to the test agent. An increase in BH3 peptide-inducedmitochondrial outer membrane permeablization in the test populationcompared to the control population indicates that the cells will beresponsive (i.e., cell death will be induced) to the test agent.Alternatively, if no change (or a decrease) in mitochondrial outermembrane permeablization in the test population compared to the controlpopulation indicates that the cells will be resistant (i.e. cell deathwill be induced) to the test agent.

The cell or cellular component is a cancer cell or a cell that issuspected of being cancerous. The cell is permeabilized to permit theBH3 peptides access to the mitochondria. Cells are permeabilized bymethods known in the art. For example, the cell are permeabilized bycontacting the cell with digitonin, or other art-recognized detergentsand cell-permeabilization agents.

After the cells are permeabilized the cells are treated with the BH3peptides or test agents. After the cell is treated, mitochondrial outermembrane permeabilization is measured. Outer membrane permeabilizationis measured by a number of methods. For example outer membranepermeabilization is measured by loss of mitochondrial membranepotential. Loss of mitochondrial membrane potential is measured forexample by treating the cells with a potentiometric or radiometric dye.

Alternatively, outer membrane permeabilization is determined bymeasuring the release of molecules from the mitochondeirialinter-membrane space. Examples of molecules that can be measured includecytochrome c and SMAC/Diablo, Omi, adenylate kinase-2 orapoptosis-inducing factor (AIF). Optionally, the cells are fixed priorto measuring outer membrane permeabilization. Cells are fixed by methodsknown in the art such as by using an aldehyde such as formaldehyde.

Mitochondial outer membrane permeabilization can be measured at thesingle cell level or multi-cell level. Additionally, some of the methodsdisclosed herein allow for subpopulations of cells to be assayed.

Examples of potentiometric dyes incude the fluorescent JC-1 probe(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide) or dihydrorhodamine 123, or tetramethylrhodamine methyl ester(TMRM) or tetramethylrhodamine ethyl ester (TMRE)

JC-1 is a lipophilic, cationic dye that enters mitochondria inproportion to the potential across the inner mitochondrial membrane.JC-1 exists as a monomer at low membrane concentrations). However, JC-1accumulates in the mitochondrial matrix under conditions of highermitochondrial potentials. At these higher concentrations, JC-1 formsred-fluorescent “J-aggregates”. As a monomer the dye has anabsorption/emission maxima of 527 nm while at high membrane potentialthe emission maximum is 590 nm. Thus, ratio measurements of the emissionof this cyanine dye can be used as a sensitive measure of mitochondrialmembrane potential. The dye allows for a dual measurement of dyeconcentration that does not require the measurement of a nuclear orcytoplasmic reference value. Studies using isolated mitochondria haveshown that the 527 nm emission from monomeric JC-1 increases almostlinearly with membrane (M) potentials ranging from 46 to 182 mV, whereasthe 590 nm J-aggregate emission is less sensitive to M values lessnegative than 140 my and is strongly sensitive to potential values inthe range of 140 to 182 mV (Di Lisa et al., 1995) Optical filtersdesigned for fluorescein and tetramethylrhodamine can be used toseparately visualize the monomer and J-aggregate forms, respectively.Alternatively, both forms can be observed simultaneously using astandard fluorescein longpass optical filter set.

Dihydrorhodamine 123 is an uncharged, nonfluorescent agent that can beconverted by oxidation to the fluorescent laser dye rhodamine 123(R123).

Release of molecules from the mitochondrial inter-membrane space can bemeasured by methods known in the art. For example, by using antibodiesto the molecules to be measured, i.e., antibodies to cytochrome c orSMAC/Diablo, Omi, adenylate kinase-2 or apoptotic-inducing factor (AIF).Detection can be for example, by ELISA, FACS, immunoblot,immunofluorescence, or immunohistochemistry.

In addition to measuring molecules that get released from themitochondrial space, other intracellular and extracellular markers canbe measured. This allows for the ability to discriminate betweensubpopulations of cells.

Dynamic BH3 profiling can be accomplished at the single cell level byimmobilizing cells on a solid surface. Optionally the solid surface ispolyamine or poly-lysine coated. Immobilized cells are permeabilized asdescribed above. The cells are then contacted with BH3 peptides and/ortest agents. After the cells have been treated for a predeterminedperiod of time such as 45-90 minutes, the cells are fixed andpermeabilized by methods known in the art. For example the cells arefixed with formaldehyde and further permeabilized with methanol ortriton x-100. Outer membrane permeabilization is determinedintracellular staining for molecules from the mitochondrialinter-membrane space and a mitrochondrial marker. Examples of moleculesthat can be measured include cytochrome c and SMAC/Diablo, Omi,adenylate kinase-2 or apoptotic-inducing factor (AIF). Mitochondrialmarkers include MnSOD. Stained cells can be counterstained with nuclearstains such as DAPI. Optionally other intracellular and extracellularmarkers can be measured. Analysis of the cells can be manuallyaccomplished using a microscope or automated for example by usingsoftware such as Cellprofiler to locate nuclei.

The cell is from a subject known to or suspected of having cancer. Thesubject is preferably a mammal. The mammal is, e.g., a human, non-humanprimate, mouse, rat, dog, cat, horse, or cow. The subject has beenpreviously diagnosed as having cancer, and possibly has alreadyundergone treatment for cancer. Alternatively, the subject has not beenpreviously diagnosed as having cancer.

The agent is a therapeutic agent such as a chemotherapeutic agent. Forexample, the agent a targeted chemotherapeutic agent such as a kinaseinhibitor. One skilled in the art will appreciate that an agent can bescreened for toxicity by the methods of the invention.

Apoptosis, i.e., cell death is identified by known methods. For example,cells shrink, develop bubble-like blebs on their surface, have thechromatin (DNA and protein) in their nucleus degraded, and have theirmitochondria break down with the release of cytochrome c, loss ofmitochondrial membrane potential, break into small, membrane-wrappedfragments, or phosphatidylserine, which is normally hidden within theinner leaflet of the plasma membrane, is exposed on the surface of thecell.

The difference in the level of mitochondrial permeabilization induced bya BH3 peptide of a cell that has been contacted with a test agentcompared to a cell that has not been contacted with the test agent isstatistically significant. By statistically significant it is meant thatthe alteration is greater than what might be expected to happen bychance alone. Statistical significance is determined by method known inthe art. For example statistical significance is determined by p-value.The p-value is a measure of probability that a difference between groupsduring an experiment happened by chance. (P(z≥z_(observed))). Forexample, a p-value of 0.01 means that there is a 1 in 100 chance theresult occurred by chance. The lower the p-value, the more likely it isthat the difference between groups was caused by treatment. Analteration is statistically significant if the p-value is or less than0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 orless.

Pro-Apoptic BCL-2 BH3 Domain Peptides

A Pro-apoptotic BCL-2 BH3 domain peptide is less than 195 amino acids inlength, e.g., less than or equal to 150, 100, 75, 50, 35, 25 or 15 aminoacid in length. Pro-apoptotic BCL-2 BH3 domain peptides include Bcl-2interacting mediator of cell death (BIM); BH3 interacting domain deathagonist (BID); Bcl-2-associated death promoter (BAD); NOXA; p53up-regulated modulator of apoptosis (PUMA); Bcl-2-modifying factor (BMF)and harakiri (HRK). A BH3 domain peptide include a peptide whichincludes (in whole or in part) the sequenceNH2-XXXXXXXXXXLXXXXDXXXX-COOH (SEQ ID NO:16). As used herein X may beany amino acid. Alternatively. The BH3 domain peptides include at least5, 6, 7, 8, 9, 15 or more amino acids of SEQ ID NO:16).

For example a Pro-apoptotic BCL-2 BH3 domain peptide includes thesequence of SEQ ID NO: 1-14 shown in Table 1. PUMA2A (SEQ ID NO: 15) isa negative control peptide.

TABLE 1 BIM Ac-MRPEIWIAQELRRIGDEFNA-NH2 SEQ ID NO: 1 BIMAc-MRPEIWIAQELRRIGDEFNV-NH2 SEQ ID NO: 2 BID EDIIRNIARHLAQVGDSMDRSEQ ID NO: 3 BIM AV MRPEIWIAQELRRIGDEFNA SEQ ID NO: 4 BID mutEDIIRNIARHAAQVGASMDR SEQ ID NO: 5 BAD LWAAQRYGRELRRMSDEFEGSFKGLSEQ ID NO: 6 BIK MEGSDALALRLACIGDEMDV SEQ ID NO: 7 NOXA AAELPPEFAAQLRKIGDKVYC SEQ ID NO: 8 NOXA B PADLKDECAQLRRIGDKVNLSEQ ID NO: 9 HRK SSAAQLTAARLKALGDELHQ SEQ ID NO: 10 PUMAEQWAREIGAQLRRMADDLNA SEQ ID NO: 11 BMF HQAEVQIARKLQLIADQFHRSEQ ID NO: 12 huBAD NLWAAQRYGRELRRMSDEFVDSFKK SEQ ID NO: 13 BAD mutLWAAQRYGREARRMSDEFEGSFKGL SEQ ID NO: 14 PUMA2A EQWAREIGAQARRMAADLNASEQ ID NO: 15

The BH3 domain peptides can be modified using standard modifications.Modifications may occur at the amino (N-), carboxy (C-) terminus,internally or a combination of any of the preceding. In one aspectdescribed herein, there may be more than one type of modification on thepolypeptide. Modifications include but are not limited to: acetylation,amidation, biotinylation, cinnamoylation, farnesylation, formylation,myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr),stearoylation, succinylation, sulfurylation and cyclisation (viadisulfide bridges or amide cyclisation), and modification by Cys3 orCys5. The GCRA peptides described herein may also be modified by 2,4-dinitrophenyl (DNP), DNP-lysine, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine).

Optionally, the BH3 domain peptide is attached to transduction domain. Atransduction domain compound that directs a peptide in which it ispresent to a desired cellular destination Thus, the transduction domaincan direct the peptide across the plasma membrane, e.g., from outsidethe cell, through the plasma membrane, and into the cytoplasm.Alternatively, or in addition, the transduction domain can direct thepeptide to a desired location within the cell, e.g., the nucleus, theribosome, the ER, mitochondria, a lysosome, or peroxisome.

In some embodiments, the transduction domain is derived from a knownmembrane-translocating sequence. Alternatively, the transduction domainis a compound that is known to facilitate membrane uptake such aspolyethylene glycol, cholesterol moieties, octanoic acid and decanoicacid.

For example, the trafficking peptide may include sequences from thehuman immunodeficiency virus (HIV) 1 TAT protein. This protein isdescribed in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, eachincorporated herein by reference. The BH3 domain peptide is linked tosome or all of the entire 86 amino acids that make up the TAT protein.For example, a functionally effective fragment or portion of a TATprotein that has fewer than 86 amino acids, which exhibits uptake intocells can be used. See e.g., Vives et al., J. Biol. Chem.,272(25):16010-17 (1997), incorporated herein by reference in itsentirety. A TAT peptide that includes the region that mediates entry anduptake into cells can be further defined using known techniques. See,e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989).Other sources for translocating sequences include, e.g., VP22 (describedin, e.g., WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)),Drosophila Antennapedia (Antp) homeotic transcription factor, HSV,poly-arginine, poly lysine, or non-viral proteins (Jackson et al, Proc.Natl. Acad. Sci. USA 89: 10691-10695 (1992)).

The transduction domain may be linked either to the N-terminal or theC-terminal end of the BH3 domain peptide. A hinge of two prolineresidues may be added between the transduction domain and BH3 domainpeptide to create the full fusion peptide. Optionally, the transductiondomain is linked to the BH3 domain peptide in such a way that thetransduction domain is released from the BH3 domain peptide upon entryinto the cell or cellular component.

The transduction domain can be a single (i.e., continuous) amino acidsequence present in the translocating protein. Alternatively it can betwo or more amino acid sequences, which are present in protein, but inthe naturally-occurring protein are separated by other amino acidsequences in the naturally-occurring protein.

The amino acid sequence of naturally-occurring translocation protein canbe modified, for example, by addition, deletion and/or substitution ofat least one amino acid present in the naturally-occurring protein, toproduce modified protein. Modified translocation proteins with increasedor decreased stability can be produced using known techniques. In someembodiments translocation proteins or peptides include amino acidsequences that are substantially similar, although not identical, tothat of naturally-occurring protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to translocationprotein to produce a modified protein having increased membranesolubility.

The BH3 domain peptide and the transduction domain can be linked bychemical coupling in any suitable manner known in the art. Many knownchemical cross-linking methods are non-specific, i.e.; they do notdirect the point of coupling to any particular site on the transportpolypeptide or cargo macromolecule. As a result, use of non-specificcross-linking agents may attack functional sites or sterically blockactive sites, rendering the conjugated proteins biologically inactive.

One way to to increase coupling specificity is to directly chemicallycouple to a functional group found only once or a few times in one orboth of the polypeptides to be cross-linked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized, See for example, Means and Feeney,Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N, N′-(1,3-phenylene) bismaleimide (both of whichare highly specific for sulfhydryl groups and form irreversiblelinkages); N,N′-ethylene-bis-(iodoacetamide) or other such reagenthaving 6 to 11 carbon methylene bridges (which relatively specific forsulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which formsirreversible linkages with amino and tyrosine groups). Othercross-linking reagents useful for this purpose include:p,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog ofMBS. The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, Chemistry Of ProteinConjugation And Cross-Linking, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the cross-linking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

The BH3 domain peptides and/or the transduction domain peptides can bepolymers of L-amino acids, D-amino acids, or a combination of both. Forexample, in various embodiments, the peptides are D retro-inversopeptides. The term “retro-inverso isomer” refers to an isomer of alinear peptide in which the direction of the sequence is reversed andthe chirality of each amino acid residue is inverted. See, e.g., Jamesonet al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693(1994). The net result of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence of the invention may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

Alternatively, the BH3 domain peptides and/or the transduction domainpeptides are cyclic peptides. Cyclic peptides are prepared by methodsknown in the art. For example, macrocyclization is often accomplished byforming an amide bond between the peptide N- and C-termini, between aside chain and the N- or C-terminus [e.g., with K3Fe(CN)6 at pH 8.5](Samson et al., Endocrinology, 137: 5182-5185 (1996)), or between twoamino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124(1988).

BH3 domain peptides and/or the transduction domain peptides are easilyprepared using modern cloning techniques, or may be synthesized by solidstate methods or by site-directed mutagenesis. A BH3 domain peptideand/or the transduction domain peptides may include dominant negativeforms of a polypeptide. In one embodiment, native BH3 domain peptidesand/or transduction domain peptides can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BH3 domain polypeptidesand/or transduction domain peptides are produced by recombinant DNAtechniques. Alternative to recombinant expression, BH3 domain peptidesand/or transduction domain peptides can be synthesized chemically usingstandard peptide synthesis techniques.

In various embodiments, the BH3 peptide maintains its secondardstructure, e.g. α-helical structure. Methods of helix stabilization areknown in the art.

Preferably, the BH3 peptide is a stable peptide. By “stable” it is meantthat the peptide possess stability sufficient to allow the manufactureand which maintains the integrity of the compound for a sufficientperiod of time to be useful for the purposes detailed herein. Forexample the peptides are covalently stabilized suing polar and or labilecrosslinks (Phelan et al. 1997 J. Am. Chem. Soc. 119:455; Leuc et al.2003 Proc. Nat'l. Acad. Sci. USA 100:11273; Bracken et al., 1994 J. Am.Chem. Soc. 116:6432; Yan et al. 2004 Bioorg. Med. Chem. 14:1403).Alternatively, the peptides are stabilized using the metathesis-basedapproach, which employed α,α—substituted non-natural amino acidscontaining alkyl tethers (Schafmeister et al., 2000 J. Am. Chem. Soc.122:5891; Blackwell et al. 1994 Angew. Chem. Int. Ed. 37:3281).Preferably the peptides are stabilized using hydrocarbon stapling.Stapled peptides are chemically braced or “stapled” peptides so thattheir shape, and therefore their activity, is restored and/ormaintained. Stably cross-linking a polypeptide having at least twomodified aminoi acids (a process termed “hydrocarbon stapling”) can helpto conformationally bestow the native secondary structure of thatpolypeptide. For example, cross-linking a polypeptide predisposed tohave an alpha-helical secondary structure can constrain the polypeptideto its native alpha-helical conformation. The constrained secondarystructure can increase resistance of the polypeptide to proteolyticcleavage and also increase hydrophobicity. Stapled CH3 peptides areproduced for example, as described in WO05044839A2, herein incorporatedby reference in its entirety. Alternatively, the BH3 peptides are cyclicpeptides. Cyclic peptides are prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K3Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124 (1988).

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the BH3domain peptide is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of BH3peptides and/or transduction domain peptides in which the protein isseparated from cellular components of the cells from which it isisolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of BH3domain peptides and/or the transduction domain peptides having less thanabout 30% (by dry weight) of non-BH3 domain peptide and/ornon-transduction domain peptides (also referred to herein as a“contaminating protein”), more preferably less than about 20% of non-BH3peptide and/or non-transduction domain peptides, still more preferablyless than about 10% of non-BH3 peptide and/or non-transduction domainpeptides, and most preferably less than about 5% non-BH3 domain peptideand/or non-transduction domain peptides . When the BH3 domain peptideand/or the transduction domain peptides or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BH3 domain peptides and/or thetransduction domain peptides in which the protein is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the protein. In one embodiment, the language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofBH3 domain peptides and/or transduction domain peptides having less thanabout 30% (by dry weight) of chemical precursors or non-BH3 domainpeptide and/or non-transduction domain peptides chemicals, morepreferably less than about 20% chemical precursors or non-BH3 domainpeptide and/or non-transduction domain peptides chemicals, still morepreferably less than about 10% chemical precursors or non-BH3 domainpeptide chemicals, and most preferably less than about 5% chemicalprecursors or non-BH3 domain peptide and/or non-transduction domainpeptides chemicals.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same apoptosis modulating effects, i.e., release ofcytochrome C or BAK oligomerization although not necessarily to the samedegree as the BH3 domain polypeptide deduced from sequences identifiedfrom cDNA libraries of human, rat or mouse origin or produced fromrecombinant expression symptoms.

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to sequences asindicated above for identity comparisons. Conservative amino acidchanges satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

BH3 domain peptides can also include derivatives of BH3 domain peptideswhich are intended to include hybrid and modified forms of BH3 domainpeptides including fusion proteins and BH3 domain peptide fragments andhybrid and modified forms in which certain amino acids have been deletedor replaced and modifications such as where one or more amino acids havebeen changed to a modified amino acid or unusual amino acid andmodifications such as glycosylation so long as the hybrid or modifiedform retains the biological activity of BH3 domain peptides. Byretaining the biological activity, it is meant that cell death isinduced by the BH3 polypeptide, although not necessarily at the samelevel of potency as that of the naturally-occurring BH3 domainpolypeptide identified for human or mouse and that can be produced, forexample, recombinantly. The terms induced and stimulated are usedinterchangeably throughout the specification.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a BH3domain polypeptide is replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a BH3 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened to identify mutants that retain activity.

Also included within the meaning of substantially homologous is any BH3domain peptide which may be isolated by virtue of cross-reactivity withantibodies to the BH3 domain peptide described herein or whose encodingnucleotide sequences including genomic DNA, mRNA or cDNA may be isolatedthrough hybridization with the complementary sequence of genomic orsubgenomic nucleotide sequences or cDNA of the BH3 domain peptidesherein or fragments thereof.

Kits

Also included in the invention are kits for performing BH3 Profilingusing whole cells. The kit consists of a multi-well plate containingstaining components in a mitochondrial buffer and a tube ofmitochondrial buffer for the suspension and dispensing of cells into theplate for analysis. Each well of the multi-well plate contains a mixtureof JC-1 dye, oligomycin, 2-mercaptoethanol, digitonin, and a peptide orsmall molecule at twice their final concentration. Optionally, the plateand suspension buffer tube can be frozen for later use along with thesuspension buffer tube. To use, the plate and buffer tube are thawed andbrought to room temperature. Cells are suspended in buffer, dispensedinto the wells of the plate, and analyzed in a fluorescence plate readerusing the JC-1 red fluorescence at 590 nm with excitation at 545 nm.

The invention will be further illustrated in the following non-limitingexamples.

Example 1: Dynamic BH3 Profiling Predicts Sensitivity to Gefitinib andWZ4002

We have found in vitro that dynamic BH3 profiling is effective topredict sensitivity to TKIs gefitinib (Iressa) and the irreversiblepyrimidine EGFR kinase inhibitor WZ4002 (that inhibits EGFR even whenthe T790M mutation is present) in the NSCLC cell lines PC9, parental andwith acquired resistance (Zhou et al., Nature 2009).

Three different cell lines were used: PC9, PC9 gefitinib resistant(PC9GR, including the mutation T790M) and PC9GR resistant to WZ4002(PC9WZR). As a test of our hypothesis, we asked whether cellulartoxicity at a late time point of 72 hours could be predicted by a shiftin mitochondrial priming at an early time point of 16 hours, a time wellbefore overt cellular toxicity could be observed. We treated the celllines with the two EGFR kinase inhibitors, gefitinib (1 μM) and WZ4002(100 nM), for 16 hours and performed the dynamic BH3 profiling analysis.We observed that the BH3 peptide Bim at low concentrations was optimalto observe changes in priming in this model.

Parental PC9 was sensitive to both gefitinib and WZ4002, showing anincrease in priming. PC9GR was insensitive to gefitinib, but sensitiveto WZ4002, also responding with increased priming. And finally PC9WZRwas insensitive to both drugs, although responds to combination ofkinase inhibitors (WZ4002 in combination with the MEK inhibitorCI-1040). This increase in priming corresponded very closely to celldeath observed at 72 hours (FIG. 1), and was significant (p=0.0052;two-tailed).

We observed that the BH3 peptide Bim with sequenceAc-MRPEIWIAQELRRIGDEFNA-NH2 (SEQ ID NO:1) at concentrations 0.3 or 1 μMwas optimal in order to predict cell death response to chemotherapy. Thepoint-mutated Bim AV BH3 peptide with sequenceAc-MRPEIWIAQELRRIGDEFNV-NH2 (SEQ ID NO:2) induced a similar response inthese same cell lines (FIG. 2).

We have found a similar significant correlation between dynamic BH3profiling and cell death using several therapies and several NSCLC lines(FIG. 3). Thus, this technique can be used in vitro to predictchemotherapy response in NSCLC.

Example 2: Dynamic BH3 Profiling Predicts Sensitivity to Imatinib

In order to prove its potential to predict chemotherapy response indifferent types of cancer, we also tested our hypothesis in cell linemodels of Chronic Myelogenous Leukemia (CML). First we used the murineBa/F3 cell line, parental and expressing the BCR-ABL fusion protein(p210), present in 95% patients with CML and can be effectively treatedwith the TKI Gleevec (imatinib). We treated both cells lines withimatinib 1 μM and we performed a dynamic BH3 profiling analysis. FIGS.6C and 6D)

Using this same approach as in Example 1, we analyzed two human CML celllines, K562 and Ku812, that constitutively express BCR-ABL, exposingthem to imatinib and performing the dynamic BH3 profiling analysis.(FIG. 4)

Both K562 and Ku812 cell lines, showed an increase in priming in severalpeptides used, but as observed previously in NSCLC, a good correlationwas observed between the increase in priming using Bim at lowconcentration (0.1 M) and cell death at 48 h. Thus, also for CML,dynamic BH3 profiling can be used to predict chemotherapy response

An important part of the application of this invention is the predictionof response to therapy in vivo. In FIG. 5, we show that pretreatmentanalysis of three patient chronic myelogenous leukemia sample correctlyidentifies the two that will respond and the one that will not respondto imatinib using dynamic BH3 profling.

Example 3: Dynamic BH3 Profiling Predicts Sensitivity to Multiple Agentsin Leukemia Cells

Using a variety of leukemia cells as a model, we tested the ability ofdynamic BH3 profling to identify agents that selectively cause celldeath (FIG. 6). We found that dynamic BH3 profling correctly identifieddrugs that would cause cell death across multiple drugs and cell lines.

Example 4: BH3 Profiling Predicts Clinical Response to Imatinib inPatients with Chronic Myelogenous Leukemia

An essential demonstration of the utility of Dynamic BH3 Profiling isthat it predicts clinical response in testing of actual primary patientcancer cells. In FIG. 5, we performed Dynamic BH3 Profiling on 24samples obtained from patients with CML. In FIG. 5A, we compare ourDynamic BH3 Profiling results with clinical response. In FIG. 5B, we usea receiver operating characteristic curve to demonstrate that DynamicBH3 Profiling predicts response to imatinib in CML patients with highsensitivity and specificity.

Example 5: BH3 Profiling Predicts Sensitivity to Multiple Agents AcrossMultiple Cancer Cell Lines

In FIG. 7, we use 9 agents to perform Dynamic BH3 Profiling on 5 celllines derived from hematologic malignancies using a 16 hour drugexposure. The Dynamic BH3 Profiling at 16 hours (7A) predictedcytotoxicity at 72 hours 7(B) with great statistical significance (7C).In FIG. 8, we use 9 agents to perform Dynamic BH3 Profiling on 5 celllines derived from solid tumors using a 16 hour drug exposure. TheDynamic BH3 Profiling at 16 hours (8A) predicted cytotoxicity at 72hours (8B) with great statistical significance (8C).

Example 6: IBH3: BH3 Profiling by Direct Measurement of RetainedCytochrome C by Facs

iBH3 adds a key fixation step to prior protocols for BH3 profiling. Thisproduced a better signal, increased sample stability, and improvedstaining to discriminate subsets in complex clinical samples. Primarytissue or cell cultures are dissociated into single cell suspensions,optionally stained for cell surface markers, and suspended in DTEBMitochondrial buffer (BH3 profiling in whole cells by fluorimeter orFACS. Methods. 2013 Apr 20. Epub ahead of print). The suspended cellsare then added to wells containing DTEB supplemented with digitonin (apermeabilizing agent) and either peptides or small molecules, which canbe prepared and frozen in sample tubes or plates, to allow the moleculesor peptides to access the mitochondria and allow for the free diffusionof cytochrome c out of permeabilized mitochondria and out of the cell.Cells are exposed to peptides/small molecules for period of time beforea short aldehyde fixation followed by neutralization with a Tris/Glycinebuffer. Anti-cytochrome c antibody is then added to each well as aconcentrate with saponin, fetal bovine serum, and bovine serum albuminto stain cytochrome c retained by the cells. Other antibodies tointracellular targets can be added at this time. Cells are analyzed byFACS to provide single cell measurements of cytochrome c afterperturbation with peptides or small molecules to provide diagnosticresponse signatures. In FIG. 11, iBH3 faithfully reproduces the profileof individual subpopulations within mixed populations. Samples profiledindividually (unmixed) or as a complex mixture (mixed) produce the sameprofile. This ability to discriminate subpopulations can be applied toany antigen or signal whether intra- or extracellular.

This is an improvement over ELISA based BH3 profiling because it cananalyze sub-populations within samples, and it is the only methodcapable of profiling using both extracellular and intracellular markers.Furthermore, it is capable of performing this analysis in highthroughput format and can be used with pre-made frozen test plateswithout the time sensitivity of live mitochondrial potentialmeasurements using potentiometric dyes.

Example 5: MicroBH3: Single Cell BH3 Profiling by ImmunofluorescenceMicroscopy

MicroBH3 (miBH3) is a BH3 profiling method where the measurement of themitochondrial effect of BH3 peptides have on individual cells bymicroscopy. To accomplish this, cells are immobilized on polyamine orpoly-lysine coated surfaces and treated with low concentrations ofdigitonin in a mitochondrial buffer to permeabilize the plasma membraneand grant access to the mitochondria without cell disruption. Fixedconcentrations of BH3 peptides or chemical compounds are added for afixed time, generally 45-90 min, before formaldehyde fixation andpermeabilization by methanol and/or triton x-100 for intracellularstaining of cytochrome C and a mitochondrial marker such as MnSOD.Stained cells are counterstained with nuclear stains such as DAPI, andfluorescent images are acquired in nuclear, mitochondrial, andcytochrome c channels. Automated analysis is performed using softwaresuch as Cellprofiler to locate nuclei, define regions adjacent to nucleithat have mitochondria, and then correlate the presence of cytochrome cwith the location of the mitochondria. Loss of localization indicates aloss of cytochrome c and a reaction to the peptide or compound. Thismethod allows the response of cells to BH3 peptides or compounds anddetermine their apoptotic propensity, or priming, at a single celllevel. Previous methods of analyzing mitochondrial integrity usingpotential sensitive fluorescent dyes use intact, not permeabilized,cells and cannot be used with BH3 peptides as they are not cellpermeant. Permeabilized cells treated with potential sensitive changeshape and are difficult to keep in focus for the necessary time coursesand are sensitive to timing. Fixed cells by this method can be readilystopped at the fixation step and can be analyzed weeks after acquisitionas well as readily re-analyzed if needed.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1-15. (canceled)
 16. A method of predicting sensitivity of a cell invivo to a therapeutic agent, the method comprising: a) contactingmitochondria of a test cell portion of a cancer cell population in vitrowith a BH3 domain peptide, wherein the cancer cell population comprisesprimary cancer cells of a patient, and wherein the test cell portion hasbeen contacted with a test therapeutic agent; b) measuring an amount ofBH3 domain peptide-induced mitochondrial outer membrane permeabilization(MOMP) in mitochondria of the test cell portion after contacting withthe BH3 domain peptide; and c) comparing the amount of MOMP inmitochondria of the test cell portion to an amount of MOMP inmitochondria of a control cell portion of the cancer cell population,wherein the control cell portion has not been contacted with the testtherapeutic agent, and wherein: (i) an increase in the amount of MOMP inmitochondria of the test cell portion compared to the amount of MOMP inmitochondria of the control cell portion indicates cancer cells of thepatient are sensitive to the test therapeutic agent in vivo, or (ii) adecrease or no change in the amount of MOMP in mitochondria of the testcell portion compared to the amount of MOMP in mitochondria of thecontrol cell portion indicates cancer cells of the patient are resistantto the test therapeutic agent in vivo.
 17. The method of claim 16,wherein the mitochondria of the test cell portion comprise mitochondriaisolated from cells of the test cell portion.
 18. The method of claim16, wherein the mitochondria of the test cell portion comprisemitochondria within cells of the test cell portion.
 19. The method ofclaim 18, wherein the BH3 domain peptide comprises a transduction domainthat translocates the BH3 domain peptide across plasma membranes ofcells of the test cell portion.
 20. The method of claim 18, wherein thecells of the test cell portion are permeabilized to permit the BH3domain peptide access to the mitochondria.
 21. The method of claim 20,further comprising permeabilizing the test cell portion prior tocontacting with the BH3 domain peptide.
 22. The method of claim 18,further comprising fixing the test cell portion prior to measuring theamount of MOMP.
 23. The method of claim 18, further comprisingcontacting the test cell portion with an antibody for an intracellularor extracellular marker to permit discrimination between cellsubpopulations based on expression of the intracellular or extracellularmarker.
 24. The method of claim 18, wherein the test cell portion isimmobilized on a solid surface.
 25. The method of claim 16, whereinmeasuring the amount of MOMP comprises: i) detecting emission from apotentiometric dye; or ii) detecting staining for cytochrome c.
 26. Themethod of claim 25, further comprising: i) contacting mitochondria ofthe test cell portion with the potentiometric dye; or ii) contactingmitochondria of the test cell portion with an antibody for cytochrome c.27. The method of claim 16, wherein the BH3 domain peptide is derivedfrom a BH3 domain of a BH3 interacting domain death agonist (BID), aBcl-2 interacting mediator of cell death (BIM), a Bcl-2-associated deathpromoter (BAD), a Noxa, a p53 up-regulated modulator of apoptosis(PUMA), a Bcl-2-modifying factor (BMF), or a harakiri (HRK) polypeptide.28. The method of claim 16, wherein the test therapeutic agent is achemotherapeutic agent.
 29. The method of claim 28, wherein thechemotherapeutic agent is a targeted chemotherapeutic agent.
 30. Themethod of claim 28, wherein the chemotherapeutic agent is a kinaseinhibitor.
 31. The method of claim 16, wherein the cancer cells comprisehematologic cancer cells.
 32. The method of claim 31, wherein thehematologic cancer cells are selected from the group consisting of acutemyeloid leukemia cells, chronic myelogenous leukemia cells, diffuselarge B-cell lymphoma cells, and multiple myeloma cells.
 33. The methodof claim 16, wherein the cancer cells comprise solid tumor cells. 34.The method of claim 33, wherein the solid tumor cells are selected fromthe group consisting of breast cancer cells, non-small cell lung cancercells, melanoma cells, and colon carcinoma cells.